Method of detecting eosinophil degranulation in the respiratory tract

ABSTRACT

Disclosed are methods of detecting eosinophil degranulation in the respiratory tract of subjects. Also, disclosed are methods of producing medical images of the respiratory tract of subjects. The method can include administering radiolabeled heparin to the respiratory tract of subjects, wherein the radiolabeled heparin binds to one or more eosinophil granule protein in the mucosal tissue of the respiratory tract.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/502,328, filed Jul. 3, 2019 (U.S. Pat. No. 11,065,351), which is acontinuation of U.S. application Ser. No. 15/719,827, filed Sep. 29,2017 (U.S. Pat. No. 10,376,602), which is a continuation of U.S.application Ser. No. 14/402,070, filed Nov. 18, 2014 (U.S. Pat. No.9,789,212), which is a U.S. national phase application of InternationalApplication No. PCT/US13/41595, which was filed May 17, 2013, and whichclaims the benefit of U.S. Provisional Patent Application No.61/688,650, filed on May 18, 2012, and U.S. Provisional PatentApplication No. 61/786,909, filed on Mar. 15, 2013. The content of theseearlier filed applications is hereby incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 AI09728awarded by the National Institutes of Health and CBET1125490 awarded bythe National Science Foundation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

This invention relates generally to the field of diagnostics. Thus,disclosed are compositions and methods for diagnosing and monitoringeosinophil degranulation-associated esophagitis in a subject, using aradiolabeled contrast agent administered orally to a subject.Specifically, disclosed are compositions and methods for diagnosing andmonitoring eosinophilic esophagitis in a subject, using radiolabeledheparin.

BACKGROUND

Eosinophilic esophagitis (EoE) is a chronic disease of the esophagusthat affects over 300,000 patients in the U.S. alone. Symptoms includedysphagia (difficulty swallowing liquids or solids or both, >90%), foodimpaction (solid food sticks in the esophagus, 50%), odynophagia(painful swallowing), heartburn (33%), chest pain, asthma (50%),diarrhea, and vomiting (Gonsalves, Kahrilas, Am J Gastroenterol, 2009).The disease primarily occurs in males (75%) with a mean age between 36and 42 years in westernized countries. While present in adults, thedisease can also manifest in children. The symptoms of EoE are similarto an atopic allergenic inflammatory condition of the esophagus,affecting up to 10% of adults presenting for upper endoscopy (Mackenzie,Aliment Ther Pharmacol, Gastroenterol, 2008).

Although the source or sources of this disease have not beenconclusively identified, investigators have identified severalcontributing factors. Genetic predisposition may be at work in thisdisease, at least in part, due to the increased incidence in firstdegree relatives of EoE patients relative to the general population.Environmental causes may also be important as allergens (i.e., food andaero-allergens) contribute in up to 97% of cases in children (Liacouras,Clin Gastro Hep, 2005). Fogg et al. (2003) observed worsening of EoEduring the pollination season in an allergic patient. Wang et al. (2007)subsequently identified a seasonal variation in identification andseverity of the disease in children. Furthermore, Mishra et al. (2001)determined that intranasal administration of Aspergillus fumigatus in amouse model replicated the esophageal eosinophilic infiltrate seen inEoE. However, EoE is not simply a seasonal allergy of the esophagus.Despite current treatment with swallowed aerosolized steroids, theresponse rate is little better than 50% (Konikoff, Gastroenterology,2007).

Food allergies also play an important role in both adult and pediatricEoE. Markowitz et al. (2003) found resolution of esophageal eosinophiliaafter 4 weeks of amino acid-based elemental diet in 49/51 pediatricpatients. In the largest analysis to date, Liacouras et al. (2005) founda 97% response to an elemental diet in a cohort of 160 children withEoE. However, preliminary data on an elimination diet in adults foundless robust responses than those observed in children. The six-foodelimination diet (Gonsalves et al., 2012) demonstrated improvement in78% and 33% complete resolution rate. Elemental diet in adults resultsin substantial improvement in eosinophilia after 4 weeks in 72% ofpatients (Peterson, 2013). Responses to skin prick testing in adultsundergoing food elimination diets suggest a multi-modal (IgE and non-IgEmediated) immunological process, and murine models find bothaero-allergens and food each play significant roles (Mishra, J ClinInvest, 2001).

In all cases, detection of EoE via a form of endoscopy known asesophagoduodenoscopy (EGD) remains essential. In this procedure, a smalltube with a camera on the distal end is passed into the esophagus,stomach, and first portion of the small intestine to visualize themucosal surfaces of these organs. In EoE, the inflammation occurs invarious parts of the esophagus; there is approximately equal incidencein the proximal, distal, or both portions of the esophagus beingaffected (Gangotena, Am J Gastroenterol, 2007) within cohorts, but suchinfiltrate varies in each individual with many demonstrating a lessintense infiltrate proximally. EoE also affects the luminal structure ofthe esophagus. Pronounced rings or furrows can develop into stricturesthat close off the esophagus, resulting in odynophagia, dysphagia, foodimpaction, and emergency hospital visits. The areas of inflammation arenot evenly distributed throughout an affected esophagus, as the diseaseoften presents in patches or select segments of the 25-30 cm long adultesophagus.

Although EGD is a key tool in the identification of EoE, some cases maynever present as a “ringed-esophagus” during EGD. A conclusive meanscurrently available to clinicians to positively identify EoE is todetect the presence of eosinophils in biopsy specimens. Tissue samplesmay be collected during EGD and then examined with traditionalhistological analysis to confirm or reject a case of EoE. However, thepatchy nature of the disease complicates collection of tissue samplesfor biopsy. When clinical suspicion for EoE is high, consensus practicerequires sampling at 4 to 5 sites throughout the esophagus. However,five 2 mm biopsy specimens represent less than 0.7% of the 20- to25-cm-long esophageal mucosa and might result in underdiagnosis of EoEif mucosal eosinophilia is particularly patchy. Specific diseasephenotypes (i.e., rings, lines, furrows, white spots, or plaques) aidphysicians in determining where and how many biopsies to perform basedon EGD-observed phenotypes, which are strong indicators of eosinophildensity. For example, biopsies to collect tissue samples are oftencollected from unaffected areas. For this reason, at least 4 (child) or5 (adult) biopsy specimens are required to confirm each case of EoE(Gonsalves Gastrointestinal Endosc, 2006; Shah Am J Gastroenterol,2009). Furthermore, additional biopsies are required to evaluate theeffectiveness of each treatment proposed. This repeated need forendoscopic removal of tissue poses a financial hardship for the patient,and the procedure can be painful, requiring sedation and/or anesthesia.

The key element for diagnosing EoE in a biopsy specimen is the presenceof eosinophils. Normal esophageal tissue does not contain eosinophils(Kato et al., 1998). These white blood cells were named for theiraffinity for the red dye eosin. Normally, eosinophils reside in theblood stream, stomach, small and large intestine, and lymphatic system(Kato et al., 1998) but infiltrate pathologically into the esophagus inEoE. In biopsy samples, an eosinophil can be identified as a cell 12-17μm in diameter with a bi-lobed nucleus and cytoplasmic granules stainingred with acidic dyes, for example eosin. A tissue count of eosinophilsin excess of 15 per field of view at high microscope power (greater than15 per high-powered field (hpf)) indicates EoE. Some clinical evidencesuggests that inflammation increases with eosinophil concentration.

A distinctive characteristic of eosinophils is their granules whichcomprise markedly cationic proteins, each of which is composed of a coreand a matrix. The core consists primarily of major basic protein 1(MBP-1); the matrix consists of eosinophil peroxidase (EPO) andeosinophil derived neurotoxin (EDN) (Peters et al., 1986), inter alia.MBP-1 is a highly basic (isoelectric point approaching 12) 13.8 kDaprotein with 5 unpaired cysteins that accounts for about 55% of thegranule's protein (Gleich et al., 1974; Gleich et al., 1976). It is amember of the C-type lectin family (lectins bind sugars) and has thehighest concentration in the eosinophil granule on a per molecule basis(Abu-Ghazaleh et al., 1992). EPO has the highest concentration in thegranule on a per mass basis, while EDN and ECP are members of the RNAse2 family (Gleich et al., 1986). Upon degranulation, an eosinophilreleases each of these proteins into the surrounding tissues. Of these,only MBP-1 stimulates histamine release (O'Donnell et al., 1983). MBP-1also exfoliates bronchial epithelial cells (Frigas et al., 1980) andcauses bronchial hyper-reactivity (Gundel et al., 1991), whereas bothMBP-1 and EPO provoke transient bronchial constriction (Gundel et al.,1991). These proteins are found in abundance in biopsies in eosinophilicesophagitis (Kephart, Am J Gastroenterol, 2010).

Currently, as symptoms are unable to predict the severity ofeosinophilic involvement, the only way to adequately monitor the extentand severity of the disease is through invasive upper endoscopy withbiopsy. Often, in food re-introduction and therapeutic evaluation, thisresults in several upper endoscopies per year for patients. Due to thecost, invasiveness, and discomfort experienced via this method ofmonitoring, patients become non-compliant, and subsequently the diseaseis not adequately tracked. Additionally, there is a lack of sensitivityof biopsies in detecting and understanding such a patchy disease becausebiopsies histologically characterize only <0.03% of the entireesophagus.

Despite the rapidly growing incidence of EoE, state-of-the-artdiagnostic techniques remain inadequate to fully characterize thisdisease. As such, there exists a need to develop a non-invasive,precise, and comprehensive technique to image and map the distributionof inflammation and deposition of eosinophil granule proteins. Suchtechniques will provide a tool to diagnose EoE, track disease activityin response to various treatment regimens, and obtain previouslyunreachable insight into the development and progression of EoEpathophysiology.

SUMMARY

In accordance with the purposes of this invention, as embodied andbroadly described herein, disclosed, in one aspect, is a method ofproducing a medical image of an esophagus in a subject, comprisingdetecting an eosinophil granule protein in the mucosal tissue of theesophagus in a subject, comprising administering to a subjectradiolabeled heparin under conditions wherein the radiolabeled heparincan bind to an eosinophil granule protein, and detecting a radiolabeledheparin/eosinophil granule protein complex in the mucosal tissue of theesophagus, whereby detecting the radiolabeled heparin/eosinophil granuleprotein complex in the mucosal tissue of the esophagus produces amedical image of the esophagus in the subject.

In another aspect, disclosed is a method of diagnosing eosinophilicesophagitis or eosinophilic diseases in a subject, comprising detectingan eosinophil granule protein in the mucosal tissue of the esophagus orother organs in a subject, comprising administering to a subjectradiolabeled heparin under conditions wherein the radiolabeled heparincan bind to an eosinophil granule protein, and detecting a radiolabeledheparin/eosinophil granule protein complex in the mucosal tissue of theesophagus or other organs, whereby detecting the radiolabeledheparin/eosinophil granule protein complex in the mucosal tissue of theesophagus diagnoses eosinophilic esophagitis or eosinophilic diseases inthe subject.

In another aspect, disclosed is a method of detecting a change ineosinophilic esophagitis in a subject diagnosed with eosinophilicesophagitis, comprising: a) producing a first medical image of theesophagus in a subject diagnosed with eosinophilic esophagitis accordingto the disclosed methods, b) producing a second medical image of theesophagus in the subject of step (a) according to the disclosed methods,and c) comparing the medical image of step (b) with the medical image ofstep (a), whereby detecting a change in the medical image of step (b)compared to the medical image of step (a) detects a change ineosinophilic esophagitis in the subject.

In another aspect, disclosed is a method of detecting eosinophildegranulation in a subject, comprising detecting an eosinophil granuleprotein in a subject, comprising administering to a subject radiolabeledheparin under conditions wherein the radiolabeled heparin can bind to aneosinophil granule protein, and detecting a radiolabeledheparin/eosinophil granule protein complex, whereby detecting theradiolabeled heparin/eosinophil granule protein complex detects thepresence of eosinophil degranulation in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows a 2D planar SPECT image of biopsies from active EoE (>15eosinophils (eos) per high power field (hpf) of view) and resolved EoEpatients (<15 eos/hpf) after being incubated with ^(99m)Tc-heparin.

FIG. 2 shows SPECT count of biopsies as a function of eosinophil countsfrom the same patients as shown in FIG. 1 .

FIG. 3 shows ^(99m)Tc-heparin binding affinity. Samples were preparedusing 100 mCi of ^(99m)Tc fresh tin solution heparin (150 mg). Sampleswere run through a Hi-trap G25 desalting column, and paperconfirmed >97% binding. (A) Shows the radiolabel activity of eachcollected chromatography fraction, which consisted of 30 drops andconstituted roughly 1 mL. (B) Shows absorbance of each fraction at 230nm. There were no second peaks of unbound ^(99m)Tc, and the activitypeak correlates with the absorbance peak.

FIG. 4 shows ^(99m)Tc-heparin binding to monkey esophagus biopsy tissue.(A) Shows a high level of initial activity of samples treated with MBP-1compared to control. The level of activity decreases with subsequentwashes before leveling out above control after the fourth wash. (B)Shows the effect of time on activity in samples treated with MBP-1. (C)Shows that activity within a sample reaches a saturation point between 1and 2 mM treatment with ^(99m)Tc-heparin.

FIG. 5 shows a SPECT intensity image of a sample of monkey esophagealtissue. The monkey esophagus was incubated overnight with PBS (control)or MBP-1 (treated). Following this initial control versus treatedincubation, the samples were incubated with ^(99m)Tc-heparin. Therepresentative result clearly demonstrates that ^(99m)Tc-heparinintensity is significantly higher in the MBP-1-treated monkey esophagealtissue.

FIG. 6 shows ex vivo biodistribution of orally administered^(99m)Tc-heparin as measured using a well counter at different timepoints. Mean and standard deviation of % ID/g were corrected forphysical decay of the isotope.

FIG. 7 shows SPECT/CT images of orally administered ^(99m)Tc-heparininto mice at 3, 6, 18, and 30 hours. St=stomach; In=Intestines.

FIG. 8 shows average net activity (μCi) of radiolabeling in differentorgans at each time interval that mice were sacrificed. The differentorgans tested include (A) esophagus, (B) stomach, (C) proximal largeintestine, (D) distal large intestine, and (E) small intestine. Theaverage is represented by the bullet, and the 95% confidence interval isrepresented by the error bars. Because only one data point was availablefor some time intervals, no error bars are shown.

FIG. 9 shows intravenous-based localization of eosinophil granuleproteins. Mice were injected subcutaneously with granule proteins MBP-1and EPO at six sites (A), followed by tail vein administration of^(99m)Tc-heparin, the same conjugate used for oral administration. After60 minutes (B), and 105 minutes (C-E), activity was apparent at thesubcutaneous MBP-1/EPO injection sites (arrows). The locations of MBP-1and EPO can be identified at relevant concentrations, with higherconcentration injections accumulating more of the ^(99m)Tc-heparin.

DETAILED DESCRIPTION

What is needed in the art are compositions and non-invasive methods fordiagnosing eosinophil degranulation-associated esophagitis in a subjectand for monitoring the effectiveness of treatment in the subject inorder to decrease suffering and cost and to increase subject compliance.Eosinophil degranulation-associated esophagitis is eosinophilicesophagitis (EoE). The disclosed methods can diagnose eosinophilicesophagitis in a subject by detecting the presence of eosinophil granuleproteins in the esophageal mucosal tissue. Thus, the diagnosis can bemade even when morphologically intact eosinophils cannot be found in theinflamed tissue under microscopic examination.

Thus, disclosed is the surprising discovery that anionic heparinradiolabeled with Technetium-99m (i.e., ^(99m)Tc or Tc-99m) can be usedas a contrast agent probe to localize cationic eosinophil granuleproteins, which are absent in the normal esophagus but are deposited inand are associated with inflammation in the mucosal tissue of theesophagus after eosinophil degranulation in a subject with EoE.

The present invention may be understood more readily by reference to thefollowing detailed description of various aspects of the invention andthe Examples included therein and to the Figures and their previous andfollowing description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods or specificradiolabeled contrast agents, as such may, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a radiolabeledcontrast agent” or a “radiolabeled heparin/eosinophil granule proteincomplex” can include mixtures of radiolabeled contrast agents ormixtures of radiolabeled heparin/eosinophil granule protein complexes,respectively, and the like.

Ranges may be expressed herein as from “about” one particular valueand/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant,both in relation to the other endpoint and independently of the otherendpoint.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a radiolabeled contrast agent is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules including the radiolabel and contrast agent are discussed,each and every combination and permutation of the radiolabel andcontrast agent and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C is disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule A-D isdisclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F isspecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-groups of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in the methods of making and usingthe disclosed radiolabeled contrast agents and radiolabeledheparin/eosinophil granule protein complexes. Thus, if there are avariety of additional steps that can be performed, it is understood thateach of these additional steps can be performed with any specific aspector combination of aspects of the disclosed methods and that each suchcombination is specifically contemplated and should be considereddisclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificaspects of the methods and compositions described herein. Suchequivalents are intended to be encompassed by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed methods and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the disclosed methods andcompositions, the particularly useful methods, devices, and materialsare as described.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such disclosure by virtue of prior invention. No admissionis made that any reference constitutes prior art. The discussion ofreferences states what their authors assert, and applicants reserve theright to challenge the accuracy and pertinence of the cited documents.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings. The word “comprise” and variations of the word, such as“comprising” and “comprises,” means “including, but not limited to” andis not intended to exclude, for example, other additives, components,integers or steps. “Optional” or “optionally” means that thesubsequently described event or circumstance may or may not occur andthat the description includes instances where said event or circumstanceoccurs and instances where it does not. As used herein, by “subject” ismeant an individual. A subject can be a mammal such as a primate, forexample, a human. The term “subject” includes domesticated animals suchas cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep,goats, etc.), and laboratory animals (e.g., mice, rabbits, rats,gerbils, guinea pigs, possums, etc.). As used herein, the terms“subject” and “patient” are interchangeable.

Disclosed are compositions and non-invasive methods for diagnosing EoEin a subject and for monitoring the course of the disease before,during, and after treatment of the disease. Thus, provided is a methodof producing a medical image of an esophagus in a subject, comprisingdetecting an eosinophil granule protein in the mucosal tissue of theesophagus in a subject, comprising administering to a subjectradiolabeled heparin under conditions wherein the radiolabeled heparincan bind to an eosinophil granule protein, and detecting a radiolabeledheparin/eosinophil granule protein complex in the mucosal tissue of theesophagus, whereby detecting the radiolabeled heparin/eosinophil granuleprotein complex in the mucosal tissue of the esophagus produces amedical image of the esophagus in the subject. In one aspect, themedical image can be three-dimensional. In another aspect, the medicalimage can be two-dimensional.

As used herein, a “mucosal tissue” is a tissue lining various cavitieswithin the body. Examples of a mucosal tissue include, but are notlimited to, mucosal tissue lining the nose, sinuses, bronchi, lungs,conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenum,jejunum, ileum, ascending colon, caecum, appendix, transverse colon,descending colon, rectum, anus, urethra, and urinary bladder. A mucosaltissue comprises an epithelial surface, glandular epithelium whichsecretes mucus, basement membrane, and submucosa with connective tissue.Thus, a radiolabeled heparin/eosinophil granule protein complex can bedetected on the epithelial surface, in the glandular epithelial tissue,on or in the basement membrane, and in the submucosal connective tissueof a mucosal tissue in a subject. In one aspect, a mucosal tissue isfrom the esophagus of a subject.

As used herein, an “eosinophil granule protein” is a protein thatcomprises the granules in eosinophils. When an eosinophil is activated,granule proteins are released from the cell into the surrounding tissue.The released granule proteins can cause pathologic allergenicinflammatory responses in the surrounding tissue, for example esophagealmucosal tissue. Examples of eosinophil granule proteins include, but arenot limited to, major basic protein (MBP), major basic protein 1(MBP-1), major basic protein 2 (MBP-2), eosinophil derived neurotoxin(EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase(EPO). Other examples of eosinophil granule proteins are provided inKita et al., Biology of Eosinophils, Chapter 19 of Immunology, which ishereby incorporated by reference for its teaching of examples ofeosinophil granule proteins. In one aspect, an eosinophil granuleprotein can be MBP-1.

As used herein, a “radiolabel” is an isotopic composition that can beattached to a substance, for example heparin, to track the substance asit passes through a system or tissue. A non-limiting example of aradiolabeled substance is radiolabeled heparin. In one aspect, aradiolabeled heparin can be ^(99m)Tc-heparin. Examples of otherradiolabels include, but are not limited to, ¹¹¹In, ¹⁴C, ³H, ¹³N, ¹⁸F,⁵¹Cr, ¹²⁵I, ¹³³Xe, ^(81m)Kr, and ¹³¹I. Other radiolabels that can beattached to a substance, for example heparin, can be found in Table 1. Aradiolabel, for example, ^(99m)Tc, can be attached to a substance, forexample heparin, using commercially available reagents well known topersons of ordinary skill in the art. In one aspect, ^(99m)Tc-heparincan be prepared as shown in Example 1 below.

TABLE 1 Commonly utilized radiolabels Nuclide Physical half-life ³H 12.3years ¹¹C 20.4 minutes ¹³N 10 minutes ¹⁴C 5730 years ¹⁵O 2 minutes ¹⁸F110 minutes ³²P 14.3 days ⁵¹Cr 27.7 days ⁵²Fe 8.3 hours ⁵⁷Co 271 days⁵⁸Co 71 days ⁵⁹Fe 45 days ⁶⁰Co 5.2 years ⁶²Zn 9.3 hours ⁶²Cu 9.7 minutes⁶⁴Cu 12.7 hours ⁶⁷Cu 2.6 days ⁶⁷Ga 78.2 hours ⁶⁸Ga 68 minutes ⁷⁶Br 16hours ^(81m)Kr — ⁸²Rb 75 seconds ⁸²Sr 25.5 days ⁸⁶Y 14.74 hours ⁸⁹Zr3.27 days ⁸⁹Sr 50.6 days ⁹⁰Sr 28.5 years ⁹⁰Y 2.7 days ⁹⁹Mo 66 hours^(99m)Tc 6.0 hours ¹¹¹In 2.8 days ¹¹³In 100 minutes ¹²³I 13.2 hours ¹²⁴I4.2 days ¹²⁵I 60 days ¹³¹I 8.0 days ¹³³Xe 5.3 days ¹³⁷Cs 30 years ¹⁵³Sm1.9 days ¹⁸⁶Re 3.8 days ²⁰¹Tl 73 hours

In one aspect, a radiolabeled contrast agent, for example^(99m)Tc-heparin, can be administered to a subject orally or byintravenous injection. Oral dosing can entail ingestion similar toroutine barium studies of the esophagus. A radiolabeled contrast agentcan be suspended in a thickened mixture (i.e., sucralose). Examples ofthickening agents include, but are not limited to, dietary starches,such as agar-agar, alginate, carrageenan, cassia gum, cellulose gum,gellan gum, guar gum, hydroxypropylcellulose, konjac gum, locust beangum, methylcellulose, hydroxypropyl methylcellulose, microcrystallinecellulose, pectin, and xanthan gum. Other viscosifiers include honey,agave nectar, date nectar, Kuzu or Kudzu root, arrow root, corn syrup,thick juices, maple syrup, coconut oil, and palm oil.

The dwell time in the esophagus can be controlled by varying theviscosity of a contrast agent and by increasing the time intervalbetween swallows, thereby providing more time for a contrast agent tocontact and bind to an eosinophil granule protein. Further, having asubject lie down with head below feet, so that there is some refluxwithin the esophagus, can prolong contact between a contrast agent andthe mucosal tissue of the esophagus in a subject.

A radiolabeled contrast agent can be administered to a subject in avolume from about 0.5 mL to about 1,000 mL, including all volumes inbetween 0.5 mL and 1,000 mL. A person of skill can determine by methodswell known in the art the volume of a contrast agent to be administeredto a subject based on the age, sex, weight, and overall condition of asubject. For example, in one aspect, the volume of a contrast agentadministered to a subject can be from about 0.5 mL to about 5 mL. Inanother aspect, the volume of a contrast agent administered to a subjectcan be from about 5 mL to about 250 mL. In another aspect, the volume ofa contrast agent administered to a subject can be from about 10 mL toabout 125 mL. In another aspect, the volume of a contrast agentadministered to a subject can be from about 15 mL to about 100 mL. Thus,the volume of a contrast agent that can be administered to a subject canbe, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 mL, and all volumes in between.

A person of ordinary skill in the art can administer to a subject acomposition comprising one or more radiolabeled contrast agents toenhance the detection of eosinophil granule proteins in the mucosaltissue of the esophagus in a subject. For example, such a compositioncan comprise ^(99m)Tc-heparin, ¹¹¹In-heparin, or ¹⁴C-heparin, or anycombination thereof. In one aspect, a radiolabeled contrast agent can be^(99m)Tc-heparin. Examples of radiolabeled heparin/eosinophil granuleprotein complexes include, but are not limited to,^(99m)Tc-heparin/MBP-1, ^(99m)Tc-heparin/MBP, ^(99m)Tc-heparin/MBP-2,^(99m)Tc-heparin/EDN, ^(99m)Tc-heparin/ECP, and ^(99m)Tc-heparin/EPO.

After administering to a subject a composition comprising radiolabeledheparin, for example ^(99m)Tc-heparin, a person of skill can use one ormore technologies and processes to detect radiolabeledheparin/eosinophil granule protein complexes in the mucosal tissue ofthe esophagus in a subject, where eosinophils have degranulated andcaused one or more patches of inflammation, to create a medical image tomap the distribution of inflammation and deposition of eosinophilgranule proteins to study the anatomy and/or pathophysiology ofeosinophilic esophagitis. Examples of technologies that can be used tocreate a medical image include, but are not limited to, single photonemission computed tomography (SPECT), positron emission (PET) scans,X-ray, conventional or computed tomography (CT), a combination of SPECTand CT, or magnetic resonance imaging (MRI). In one aspect, for example,SPECT can optionally be used in combination with MRI and/or CT scans toproduce a medical image of an esophagus having patches of eosinophilicesophagitis. Fiduciary markers on the skin of a subject can also be usedto position a subject so that the subject can be imaged from day to day.For example, lasers can be used to position a subject reproducibly. Thispermits use of multiple scans to be precisely compared. In one aspect, amedical image can be three-dimensional. In another aspect, a medicalimage can be two-dimensional.

Also disclosed is a method of diagnosing eosinophilic esophagitis in asubject, comprising detecting an eosinophil granule protein in themucosal tissue of the esophagus in a subject, comprising administeringto a subject radiolabeled heparin under conditions wherein theradiolabeled heparin can bind to an eosinophil granule protein, anddetecting a radiolabeled heparin/eosinophil granule protein complex inthe mucosal tissue of the esophagus, whereby detecting the radiolabeledheparin/eosinophil granule protein complex in the mucosal tissue of theesophagus diagnoses eosinophilic esophagitis in the subject. In oneaspect, a radiolabeled heparin/eosinophil granule protein complex can be^(99m)Tc-heparin/MBP-1.

In one aspect, a radiolabeled contrast agent, for example^(99m)Tc-heparin, can be administered to a subject orally. Oral dosingcan entail ingestion similar to routine barium studies of the esophagus.A radiolabeled contrast agent can be suspended in a thickened mixture(i.e., sucralose). Examples of thickening agents include, but are notlimited to, dietary starches, such as agar-agar, alginate, carrageenan,cassia gum, cellulose gum, gellan gum, guar gum, hydroxypropylcellulose,konjac gum, locust bean gum, methylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, pectin, and xanthan gum.Other viscosifiers include honey, agave nectar, date nectar, Kuzu orKudzu root, arrow root, corn syrup, thick juices, maple syrup, coconutoil, and palm oil.

The dwell time in the esophagus can be controlled by varying theviscosity of a contrast agent and by increasing the time intervalbetween swallows, thereby providing more time for a contrast agent tocontact and bind to an eosinophil granule protein. Further, having asubject lie down with head below feet, so that there is some refluxwithin the esophagus, can prolong contact between a contrast agent andthe mucosal tissue of the esophagus in a subject.

A radiolabeled contrast agent can be administered to a subject in avolume from about 0.5 mL to about 1,000 mL, including all volumes inbetween 0.5 mL and 1,000 mL. A person of skill can determine by methodswell known in the art the volume of a contrast agent to be administeredto a subject based on the age, sex, weight, and overall condition of asubject. For example, in one aspect, the volume of a contrast agentadministered to a subject can be from about 0.5 mL to about 5 mL. Inanother aspect, the volume of a contrast agent administered to a subjectcan be from about 5 mL to about 250 mL. In another aspect, the volume ofa contrast agent administered to a subject can be from about 10 mL toabout 125 mL. In another aspect, the volume of a contrast agentadministered to a subject can be from about 15 mL to about 100 mL. Thus,the volume of a contrast agent that can be administered to a subject canbe, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 mL, and all volumes in between.

A person of ordinary skill in the art can administer to a subject acomposition comprising one or more radiolabeled contrast agents toenhance the detection of eosinophil granule proteins in the mucosaltissue of the esophagus in a subject. For example, such a compositioncan comprise ^(99m)Tc-heparin, ¹¹¹In-heparin, or ¹⁴C-heparin, or anycombination thereof. In one aspect, a radiolabeled contrast agent can be^(99m)Tc-heparin. Examples of radiolabeled heparin/eosinophil granuleprotein complexes include, but are not limited to,^(99m)Tc-heparin/MBP-1, ^(99m)Tc-heparin/MBP, ^(99m)Tc-heparin/MBP-2,^(99m)Tc-heparin/EDN, ^(99m)Tc-heparin/ECP, and ^(99m)Tc-heparin/EPO.

After administering to a subject a composition comprising radiolabeledheparin, for example ^(99m)Tc-heparin, a person of skill can use one ormore technologies and processes to detect radiolabeledheparin/eosinophil granule protein complexes in the mucosal tissue ofthe esophagus in a subject, where eosinophils have degranulated andcaused one or more patches of inflammation, to create a medical image tomap the distribution of inflammation and deposition of eosinophilgranule proteins to study the anatomy and/or pathophysiology ofeosinophilic esophagitis. Examples of technologies that can be used tocreate a medical image include, but are not limited to, single photonemission computed tomography (SPECT), positron emission (PET) scans,X-ray, conventional or computed tomography (CT), a combination of SPECTand CT, or magnetic resonance imaging (MRI). In one aspect, for example,SPECT can optionally be used in combination with MRI and/or CT scans toproduce a medical image of an esophagus having patches of eosinophilicesophagitis. Fiduciary markers on the skin of a subject can also be usedto position a subject so that the subject can be imaged from day to day.For example, lasers can be used to position a subject reproducibly. Thispermits use of multiple scans to be precisely compared. In one aspect, amedical image can be three-dimensional. In another aspect, a medicalimage can be two-dimensional.

Further disclosed is a method of detecting a change in eosinophilicesophagitis in a subject diagnosed with eosinophilic esophagitis,comprising: (a) producing a first medical image of the esophagus in asubject diagnosed with eosinophilic esophagitis according to thedisclosed methods, (b) producing a second medical image of the esophagusin the subject of step (a) according to the disclosed methods, and (c)comparing the medical image of step (b) with the medical image of step(a), whereby detecting a change in the medical image of step (b)compared to the medical image of step (a) detects a change ineosinophilic esophagitis in the subject. In one aspect, the medicalimage can be three-dimensional. In another aspect, the medical image canbe two-dimensional.

Thus, a person of skill can produce a first medical image of theesophagus in a subject diagnosed with EoE to have as a baseline forfuture comparison with later-produced medical images of the esophagus inthe subject to determine what the natural history of EoE is. Further, afirst medical image can be used to determine whether a treatment of EoEis effective in the subject. For example, if a second medical image isproduced after the initiation of treatment of EoE in a subject and thesecond medical image shows fewer areas of radiolabeledheparin/eosinophil granule protein complexes (i.e., inflammation) whencompared to the first medical image produced before initiation oftreatment, a person of skill, for example a physician, can determinethat the treatment of EoE in the subject is effective. Conversely, if asecond medical image is produced after the initiation of treatment ofEoE in a subject and the second medical image shows the same or moreareas of radiolabeled heparin/eosinophil granule protein complexes(i.e., inflammation) when compared to the first medical image producedbefore initiation of treatment, a person of skill, for example aphysician, can determine that the treatment of EoE in the subject is noteffective.

In another aspect, disclosed is a method of detecting eosinophildegranulation in a subject, comprising detecting an eosinophil granuleprotein in a subject, comprising administering to a subject radiolabeledheparin under conditions wherein the radiolabeled heparin can bind to aneosinophil granule protein, and detecting a radiolabeledheparin/eosinophil granule protein complex, whereby detecting theradiolabeled heparin/eosinophil granule protein complex detectseosinophil degranulation in the subject.

In one aspect, a radiolabeled contrast agent, for example^(99m)Tc-heparin, can be administered to a subject orally orintravenously. Oral dosing can entail ingestion similar to routinebarium studies of the esophagus. A radiolabeled contrast agent can besuspended in a thickened mixture (i.e., sucralose). Examples ofthickening agents include, but are not limited to, dietary starches,such as agar-agar, alginate, carrageenan, cassia gum, cellulose gum,gellan gum, guar gum, hydroxypropylcellulose, konjac gum, locust beangum, methylcellulose, hydroxypropyl methylcellulose, microcrystallinecellulose, pectin, and xanthan gum. Other viscosifiers include honey,agave nectar, date nectar, Kuzu or Kudzu root, arrow root, corn syrup,thick juices, maple syrup, coconut oil, and palm oil.

The dwell time in the esophagus can be controlled by varying theviscosity of a contrast agent and by increasing the time intervalbetween swallows, thereby providing more time for a contrast agent tocontact and bind to an eosinophil granule protein. Further, having asubject lie down with head below feet, so that there is some refluxwithin the esophagus, can prolong contact between a contrast agent andthe mucosal tissue of the esophagus in a subject.

A radiolabeled contrast agent can be administered to a subject in avolume from about 0.5 mL to about 1,000 mL, including all volumes inbetween 0.5 mL and 1,000 mL. A person of skill can determine by methodswell known in the art the volume of a contrast agent to be administeredto a subject based on the age, sex, weight, and overall condition of asubject. For example, in one aspect, the volume of a contrast agentadministered to a subject can be from about 0.5 mL to about 5 mL. Inanother aspect, the volume of a contrast agent administered to a subjectcan be from about 5 mL to about 250 mL. In another aspect, the volume ofa contrast agent administered to a subject can be from about 10 mL toabout 125 mL. In another aspect, the volume of a contrast agentadministered to a subject can be from about 15 mL to about 100 mL. Thus,the volume of a contrast agent that can be administered to a subject canbe, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 mL, and all volumes in between.

A person of ordinary skill in the art can administer to a subject acomposition comprising one or more radiolabeled contrast agents toenhance the detection of eosinophil granule proteins in the mucosaltissue of the esophagus in a subject. For example, such a compositioncan comprise ^(99m)Tc-heparin, ¹¹¹In-heparin, or ¹⁴C-heparin, or anycombination thereof. In one aspect, a radiolabeled contrast agent can be^(99m)Tc-heparin. Examples of radiolabeled heparin/eosinophil granuleprotein complexes include, but are not limited to,^(99m)Tc-heparin/MBP-1, ^(99m)Tc-heparin/MBP, ^(99m)Tc-heparin/MBP-2,^(99m)Tc-heparin/EDN, ^(99m)Tc-heparin/ECP, and ^(99m)Tc-heparin/EPO.

After administering to a subject a composition comprising radiolabeledheparin, for example ^(99m)Tc-heparin, a person of skill can use one ormore technologies and processes to detect radiolabeledheparin/eosinophil granule protein complexes in the mucosal tissue ofthe esophagus in a subject, where eosinophils have degranulated andcaused one or more patches of inflammation, to create a medical image tomap the distribution of inflammation and deposition of eosinophilgranule proteins to study the anatomy and/or pathophysiology ofeosinophilic esophagitis. Examples of technologies that can be used tocreate a medical image include, but are not limited to, single photonemission computed tomography (SPECT), positron emission (PET) scans,X-ray, conventional or computed tomography (CT), a combination of SPECTand CT, or magnetic resonance imaging (MRI). In one aspect, for example,SPECT can optionally be used in combination with MRI and/or CT scans toproduce a medical image of an esophagus having patches of eosinophilicesophagitis. Fiduciary markers on the skin of a subject can also be usedto position a subject so that the subject can be imaged from day to day.For example, lasers can be used to position a subject reproducibly. Thispermits use of multiple scans to be precisely compared. In one aspect, amedical image can be three-dimensional. In another aspect, a medicalimage can be two-dimensional.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight; temperature is in ° C. or is atambient temperature; and pressure is at or near atmospheric.

Example 1 Preparation of ^(99m)Tc-Heparin

Solutions of stannous chloride (40 mg/mL, Sigma 243523) were prepared indeionized water under flowing nitrogen. A 0.5 mL aliquot was filteredand mixed with 1.00 mL NaCl (1.00 M) plus 150 mg of preservative-freeheparin (10,000 IU/mL). Approximately 100 mCi of freshly eluted ^(99m)Tcwas added and mixed for 30 minutes at room temperature. Aliquotscontaining approximately 10 mCi of ^(99m)Tc and 20 mg of heparin wereremoved for tissue experiments. FIG. 3A shows the labeling affinity,measured by paper chromatography Whatman number 31 with acetone, showedmore than 97% binding of heparin to ^(99m)Tc.

Radiolabeled heparin was also analyzed by Sephadex G25 columnchromatography (HiTrap 5 mL desalting columns, GE healthcare, 17140801)with 0.15 M NaCl as the elution buffer, and approximately 1 mL fractionswere collected. FIG. 3B shows radioactivity eluted at fraction 4 (free^(99m)Tc elutes at fraction 13), demonstrating that all of the ^(99m)Tceluted at the void volume and confirming that there is no unbound^(99m)Tc in the radiolabeled heparin. The stability of ^(99m)Tc-heparinin an acidic environment was tested by diluting in artificial gastricjuice (Carolina, 864603) and showing that its properties were unaltered,using both paper chromatography and Sephadex G25.

Example 2 Esophageal Biopsies

Esophageal biopsies were collected from a prior elemental diet study(IRB 00040035 University of Utah) and kept frozen at −70° C. Biopsiesfrom each patient were selected for histological analysis for eosinophildensity or radiolabeling. Biopsies from EoE patients who resolved theireosinophilia under the elemental diet were chosen as negative controls.Tissues from resolved EoE and active EoE patients were incubated withthe ^(99m)Tc-heparin, washed in buffered saline, and imaged using SPECT.

Example 3 Analysis of Radiolabeled Biopsies

The activity of radiolabeled biopsy tissues was measured using dosecalibrators (Capintec model CRC-15R). Two-dimensional planar staticimages of the radiolabeled biopsies were obtained using the Ecam SPECTinstrument (Siemens). The images were acquired for 3 minutes each usinga 1.0 magnification, 256 by 256 matrix size. Correlation betweeneosinophil densities and SPECT counts obtained by 2D imaging wereevaluated by Spearman rank correlation analysis.

Table 2 presents the SPECT counts of each biopsy, summarizing itsradioactivity and eosinophil density. Histopathological analysesconfirmed that only samples 1-5 had greater than 15 eosinophils/hpf.FIGS. 1 and 2 show that active EoE cases can be differentiated fromresolved EoE cases using this technique. The Spearman rank correlationanalysis found a monotonic correlation between eosinophil density andSPECT count with a p value less than 0.05. Even though the eosinophildensities were not derived from identical biopsy tissues used for SPECTimaging, tissues from inflamed patients displayed significant binding to^(99m)Tc-heparin that correlates with eosinophil density.

TABLE 2 Radioactivity measurements, histological data, and the SPECTcount of EoE versus control samples Sample Disease Eosinophil ActivitySPECT Number Criteria Density/hpf μCi Count 1 Active EoE 68 180 59634 2Active EoE 65 128 40474 3 Active EoE 21 101 46457 4 Active EoE 23 13159923 5 Active EoE 21 70 31780 6 Resolved EoE 10 147 47048 7 ResolvedEoE 1 29.3 13202 8 Resolved EoE 1 37 17518

Example 4 Quality Control of Radiolabeled Heparin

Thin layer chromatography (TLC) can be performed on Whatman 31ET stripswith acetone as the solvent. A chromatography chamber can be prepared byadding just enough acetone solvent so that it covers the bottom of thechromatography chamber. A small sample of ^(99m)Tc-heparin can be placedat 1 cm from the bottom of the strip. This is the origin. A spotted SGstrip can be placed into the chromatography chamber making sure that theorigin of the sample is not immersed in the solvent. When the solventreaches the top of the strip, the solvent front, the strip can beremoved from the chromatography chamber. The strip can be cut in halfbetween the origin and solvent front. Each strip can be counted usingthe Ludlum 2200 scaler/ratemeter attached to a 1″×1″ sodium iodidedetector. Each strip can be counted for 1 minute (cpm). A background(bkg) count can be obtained before counting each strip. The backgroundcount can be subtracted from each count. The radiochemical purity for^(99m)Tc-heparin can be calculated using the following formula:

Background cpm (bkg), Origin cpm, and Solvent Front cpm are measured.

${{Percentage}{\,^{99m}{Tc}}}‐{{heparin} = \frac{\left( {{{Origin}{cpm}} - {{bkg}{cpm}}} \right)}{\left( {{{Origin}{cpm}} - {{bkg}{cpm}}} \right) + \left( {{{Solvent}{Front}{cpm}} - {{bkg}{cpm}}} \right)}}$

The final product can be clear and free of particulate matter. Theradiochemical purity for ^(99m)Tc-heparin can be >90%.

Example 5 Alternative Preparation of ^(99m)Tc-Heparin

^(99m)Tc-heparin was prepared by diluting stannous chloride (5 mg/mL,Sigma 243523) in water under flowing nitrogen. A 0.10 mL aliquot wasfiltered and mixed with 15 mg of preservative free heparin (10,000IU/mL). Approximately 5 mCi of freshly eluted ^(99m)Tc was added andmixed in for 15 minutes at room temperature. Hundred microliter aliquotscontaining approximately 0.5 mCi of ^(99m)Tc and 1.5 mg of heparin wereremoved for oral administration to mice. Quality control was determinedby paper chromatography (Whatman no. 31) with acetone.

Example 6 Quantitative Organ Biodistribution

All animal experiments were performed in accordance with approvedUniversity of Utah Institutional Animal Care and Use Committee (IACUC)protocols. ^(99m)Tc-heparin (approximately 0.5 mCi/mouse, 15 mg) wasadministered directly to the stomach of healthy adult mice (C57Bl/6,Charles River Laboratories International, Inc., average weight=29.14±1.5g) by oral gavage under brief restraint using a straight 1.5 in.,20-gauge needle with a 1.25 mm ball tip. Three animals per group wereeuthanized with CO₂ at 45 minutes, 1.5, 3, 6, 18, and 30 hours. Withcare to avoid cross-contamination, whole organs, blood, and hind legmuscle samples were harvested within approximately 20 min, weighed, andcounted for up to 2 min in captus 3000 well counter (Bicorn model2MW2/2-X NaI drilled well crystal detector in a Canberra 727 well). Theproximal large intestine was considered to include the caecum plus ¾ indistal small intestine, while the remainder was considered distal largeintestine. Larynx and thyroid were harvested and measured together. Nogastrointestinal pelvic samples included reproductive organs andsurrounding fatty tissue. Results are expressed as the percentageinjected dose per gram of harvested organ (% ID/g).

The biodistribution of ^(99m)Tc-heparin acquired at time of harvest asmeasured by well counting is shown in FIG. 6 . Forty-five minutes afteroral administration, the stomach and small intestine were the majororgans of ^(99m)Tc-heparin accumulation. The proximal large intestineshowed the highest uptake at 1.5 h after oral administration. Themajority of activity after 6 h incubation of ^(99m)Tc-heparin was indistal large intestine. Little uptake was observed in esophagus, lungand thyroid. The very modest uptake in the esophagus may be due toesophageal reflux associated with the oral gavage. All other organsshowed negligible uptake in all the time points, indicating that^(99m)Tc-heparin is not absorbed significantly outside of the GI tract.

Example 7 SPECT Imaging

To obtain qualitative images of ^(99m)Tc activity, mice wereanesthetized with isoflurane gas (5% induction, 1-2% maintenance,IMWI/VetOne, Meridian, Id., Cat #501017) and positioned prone on thescanner bed 40 min prior to euthanasia time. SPECT/CT images of micewere acquired by using an INVEON™ trimodality PET/SPECT/CT scanner(Siemens Preclinical Solutions, Knoxville, Tenn.). A sensor (Biovet,France) was used to monitor the respiration rate of mice underanesthesia. CT images consisting of 220 degrees and 480 projections ateach of 2 bed positions were acquired first. The exposure time was 135ms with a detector setting at 80 peak kilovoltage (kVp) and 500 μA. Datawere reconstructed onto a 416×416×752 image matrix using the COBRAsoftware package (Exxim Computing Corporation, Pleasanton, Calif.). Theeffective image pixel size was 97 μm. SPECT data were acquiredimmediately following the CT using a 2 mm single pinhole collimator witha detector radius of rotation at 35 mm. Images were acquired over 1.5detector revolutions with 6° between each of 90 projections. A 90 mm bedtravel was used. Each projection was acquired for 12 seconds, and thedata were histogrammed with a 10% window centered at 140 keV.Reconstruction was performed using ordered subset expectationmaximization 3D (OSEM3D) with 8 iterations and 6 subsets. Reconstructedimages were analyzed and visualized using the

Siemens INVEON™ Research Workplace (IRW) Software.

To visually track the radiolabeled dose in mice, qualitative images of^(99m)Tc activity were obtained with SPECT/CT. SPECT/CT images of micewere acquired at 3, 6, 18, and 30 hours after oral administration. FIG.7 shows the activity mostly in the GI tract, i.e., stomach andintestines. No localization was visualized in liver, kidney, or lung,indicating that ^(99m)Tc-heparin was not absorbed through the GI tractand was not circulating in the blood.

Example 8 Radiation Dose Calculation for Orally Administered^(99m)Tc-heparin

The OLINDA/EXM dose estimation software was used to determine theeffective doses and doses to individual organs on calculations andconstants defined in the ICRP Publication 106. This software includes akinetic input module with exponential curve fitting of the input datafor determining the number of disintegrations per unit of administeredactivity (μCi-h/μCi) in each source organ. Residence times in thevarious organs were measured using trapezoidal averaging of the averageddata for each time interval.

Radiation dose calculations were performed based on the data obtainedfrom mice administered ^(99m)Tc-heparin by oral gavage. The data wereobtained from 18 mice sacrificed at six time intervals, with a total ofthree data points at each time interval. Dose calculations wereperformed based on the simple average of the three data points at eachtime interval. The 95% confidence interval was calculated based on thesedata points.

Good recovery of the ^(99m)Tc-heparin was observed. Between 89 and 99%of the administered material and 99% of the material measured just priorto sacrifice was recovered.

Residence times in the various organs were measured using trapezoidalaveraging of the averaged data for each time interval. A simple averageof the data was used.

Mouse to human estimate was performed through simple mass weightingtechniques. Human data were based on ICRP 106 values. Mouse data weremeasured for each mouse and individual organ. Simple averages ofmouse-to-human estimates were used.

Data were collected for 22 different organs. However, over 98% of theaccumulated activity was observed in the GI tract. FIG. 8 shows theaverage net activity (μCi) at each time interval. The uptake by theproximal and distal intestine gradually increased over 3 and 6 hours,respectively, and then decreased until 30 hours. Little radioactivitylevel was observed in esophagus, lung and thyroid, and negligibleactivity was observed elsewhere.

Using the average accumulated activities (scaled for mouse-to-humanestimate), the average dose to individual organs was calculated. Table 3shows the results of the estimated dose to a human from orallyadministered ^(99m)Tc-heprin. These results are listed in descendingorder based on the dose to the organ. Note that greater than 98% of theorgan dose is to the organs of the GI tract.

TABLE 3 Dose to individual organ from orally administered^(99m)Tc-heparin, human corrected Human corrected Fraction Organ A(μCi-hr/μCi) of Total Dist. Lg. Int. 4.355E−01 2.916E−01 Small Int.4.226E−01 2.829E−01 Prox. Lg. Int. 3.602E−01 2.411E−01 Stomach 2.541E−011.701E−01 Lung 2.012E−02 1.347E−02 Urine 3.830E−04 2.564E−04 Liver2.708E−04 1.813E−04 Esophagus 2.359E−04 1.579E−04 Larynx/Thyroid1.381E−04 9.248E−05 Bladder 9.002E−05 6.026E−05 Kidney 7.918E−055.300E−05 Heart 4.281E−05 2.866E−05 Trachea 3.724E−05 2.493E−05 Pancreas2.300E−05 1.540E−05 Spleen 1.452E−05 9.719E−06 Testicles 1.206E−068.072E−07 Thymus 6.022E−07 4.032E−07 Gall Bladder 5.582E−07 3.737E−07

Having estimated residence times and organ doses, the effective dose wasestimated. In order to estimate effective dose, OLINDA/EXM doseestimation software was used. OLINDA is an FDA approved software toolfor dose estimation and has received 501(k) approval. The estimatedresidence times were input into OLIDNA. The results for adults are shownin Table 4. Note that approximately 74% of the contribution to theeffective dose is from the organs in the GI tract. The dose contributionfrom the ovaries and bone marrow was a result of the accumulatedactivity in other organs (i.e., irradiation from accumulated activity insurrounding organs). Accumulated activity in these organs was notspecifically determined.

TABLE 4 OLINDA results for Adults using estimated residence timesFraction of mGy/MBq Total Dose LLI Wall 2.46E−03 5.12E−01 Stomach Wall1.04E−03 2.17E−01 Ovaries 8.87E−04 1.85E−01 Red Marrow 1.02E−04 2.12E−02ULI Wall 6.83E−05 1.42E−02 Urinary Bladder Wall 6.64E−05 1.38E−02 Lungs4.92E−05 1.02E−02 Small Intestine 4.68E−05 9.75E−03 Liver 2.99E−056.23E−03 Uterus 1.32E−05 2.75E−03 Pancreas 8.27E−06 1.72E−03 OsteogenicCells 7.83E−06 1.63E−03 Spleen 5.61E−06 1.17E−03 Kidneys 4.84E−061.01E−03 Breasts 4.73E−06 9.85E−04 Muscle 3.00E−06 6.25E−04 Adrenals2.70E−06 5.62E−04 Skin 1.77E−06 3.69E−04 Thyroid 5.46E−07 1.14E−04Thymus 3.60E−07 7.50E−05 Brain 5.45E−09 1.13E−06 Gallbladder Wall0.00E+00 0.00E+00 Heart Wall 0.00E+00 0.00E+00 Testes 0.00E+00 0.00E+00Effective Dose 4.81E−03 (mSv/MBq)

Example 9 Patient Administration

A patient may not eat or drink anything except water after midnight thenight before the scan. An oral dose of about 10 mCi ^(99m)Tc-heparin canbe administered to the patient.

2D planar images can be acquired over time during the swallowingprocess. Additional 2D planar body images can be taken and used todetermine the relative biodistribution of ^(99m)Tc-heparin. A 3D SPECTimage can be acquired. The patient can be re-imaged after drinkingwater.

Upon completion of the study, the patient can be removed from thescanner and encouraged to void. The patient can be instructed to drinkplenty of fluids and void frequently throughout the day to help reduceradiation exposure.

Example 10 Monkey Esophagus Tissue Experiment

Monkey esophagus biopsy samples were incubated with MBP-1 (treatedsample) overnight. Samples incubated with PBS were used as a negativecontrol. Radiolabeled binding of ^(99m)Tc-heparin was examined as afunction of washing steps with PBS, time, and heparin molarity. Resultsof binding experiments are shown in FIG. 4 . FIG. 5 shows arepresentative SPECT image of monkey esophagus biopsy tissue afterincubation with ^(99m)Tc-heparin. Higher SPECT intensity of^(99m)Tc-heparin is clearly visible in the MBP-1 treated sample comparedto the control.

Example 11 Co-Localization of ^(99m)Tc-Heparin Activity with EosinophilGranule Protein Injection Sites

In order to demonstrate in vivo targeting and intravenous administrationof the radiocontrast agent ^(99m)Tc-heparin, eosinophil granule proteinsMBP-1 and EPO were injected subcutaneously into healthy, anesthetizedC57BL6 mice to simulate local eosinophilia, as a positive control.^(99m)Tc-heparin was administered intravenously via tail vein after theinjection of the eosinophil granule proteins and was given time to bindthe eosinophil granule proteins.

FIG. 9A depicts the sites and concentrations of the subcutaneouslyinjected eosinophil granule proteins. The mice were imaged using anInveon trimodality scanner (Siemens Preclinical Solutions, Knoxville,Tenn.). SPECT/CT Scans were completed at 60 and 105 minutes afterintravenous administration of ^(99m)Tc-heparin.

SPECT/CT images depicting the distribution of ^(99m)Tc-heparin are shownin FIG. 9 (B-E). The ^(99m)Tc-heparin spread throughout the major bloodorgans, particularly the liver, spleen, and kidneys, as well as thebladder. However, there was additional activity at the injection sites(arrows) not present in negative control mice that had not been injectedwith eosinophil granule proteins, indicating successful co-localizationof ^(99m)Tc-heparin with the eosinophil granule proteins. Activity wasclearly visible and correlated with the concentration of MBP-1 at theinjection sites. Only the highest concentration EPO site clearly showedsignificant ^(99m)Tc-heparin presence. Observations at necropsy revealedno significant hemorrhage to confound the positive result.

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It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

What is claimed is:
 1. A method of detecting eosinophil degranulation ina respiratory tract passage of a subject, the method comprising:administering radiolabeled heparin to the respiratory tract passage ofthe subject, wherein the radiolabeled heparin binds to one or moreeosinophil granule proteins in the mucosal tissue of the respiratorytract passage; and detecting the radiolabeled heparin in the mucosaltissue of the respiratory tract passage by one of single-photon emissioncomputed tomography (SPECT), positron emission tomography (PET),conventional or computed tomography (CT), magnetic resonance imaging(MRI), or combinations thereof, hereby wherein detecting theradiolabeled heparin in the mucosal tissue is indicative of eosinophildegranulation in the respiratory tract passage of the subject.
 2. Themethod of claim 1, wherein the radiolabeled heparin binds to the one ormore eosinophil granule proteins to form a radiolabeled heparineosinophil granule protein complex, and wherein detecting theradiolabeled heparin comprises detecting the radiolabeled heparineosinophil granule protein complex by one of single-photon emissioncomputed tomography (SPECT), positron emission tomography (PET),conventional or computed tomography (CT), magnetic resonance imaging(MRI), or combinations thereof.
 3. The method of claim 1, wherein theone or more eosinophil granule proteins comprise one or more of majorbasic protein 1 (MBP-1), major basic protein 2 (MBP-2), eosinophilderived neurotoxin (EDN), eosinophil cationic protein (ECP), andeosinophil peroxidase (EPO).
 4. The method of claim 1, wherein theradiolabel is ^(99m)Tc.
 5. The method of claim 1, wherein therespiratory tract passage is selected from the group consisting of anose, a sinus, a lung, and a bronchus of the subject.
 6. The method ofclaim 1, wherein the respiratory tract passage is a paranasal sinuscavity.
 7. A method of producing a medical image of a respiratory tractpassage of a subject, the method comprising: administering radiolabeledheparin to the respiratory tract passage of the subject, wherein theradiolabeled heparin binds to one or more eosinophil granule proteins inthe mucosal tissue of the respiratory tract passage; and detecting theradiolabeled heparin by one of single-photon emission computedtomography (SPECT), positron emission tomography (PET), conventional orcomputed tomography (CT), magnetic resonance imaging (MRI), orcombinations thereof, thereby producing a medical image of therespiratory tract passage of the subject.
 8. The method of claim 7,wherein the radiolabeled heparin binds to the one or more eosinophilgranule proteins to form a radiolabeled heparin eosinophil granuleprotein complex and wherein detecting the radiolabeled heparin comprisesdetecting the radiolabeled heparin eosinophil granule protein complex byone of single-photon emission computed tomography (SPECT), positronemission tomography (PET), conventional or computed tomography (CT),magnetic resonance imaging (MRI), or combinations thereof.
 9. The methodof claim 7, wherein the one or more eosinophil granule proteins compriseone or more of major basic protein 1 (MBP-1), major basic protein 2(MBP-2), eosinophil derived neurotoxin (EDN), eosinophil cationicprotein (ECP), and eosinophil peroxidase (EPO).
 10. The method of claim7, wherein the radiolabel is ^(99m)Tc.
 11. The method of claim 7,wherein the respiratory tract passage is selected from the groupconsisting of a nose, a sinus, a lung, and a bronchus of the subject.12. The method of claim 7, wherein the respiratory tract passage is aparanasal sinus cavity.